Phytochemical and Quality Assessment of Acacia nilotica Linn and Acacia leucophloea willd Flowers

Raju and Leela: Phytochemical and Quality Assessment of Acacia nilotica Linn and Acacia leucophloea willd Flowers

Authors

INTRODUCTION

Herbal medicines have been used in medical practice for thousands of years and recognized especially as a valuable and readily available resource for health care. Traditional herbs and herbal products have been considered to be mild, non-toxic and even harmless because of their natural origin. Like other crops, medicinal plants are susceptible to insects and diseases both in the field and the storage, so pesticides are widely used for their protection. In fact, contamination of crude medicinal plants as well as their products has increasingly been reported.1,2 This has brought concernsand fears regarding practitioner’s professionalism and quality, efficacy and safety of their treatment methods and also for the products from herbal and natural sources available in market. Because of the high cost of pesticide’ free cultivation, organic cultivation is only possible on a small scale and wild raw material is of insufficient quantity to meet the needs to the herbal drug market. The ever increasing consumption of medicinal plants necessitates large scale cultivation of medicinal plants which is not possible without use of pesticides. Attentions usually focused on contamination by organochlorine pesticides (OCPs) due to their toxicity and persistence in environment and contamination by common pesticides.3,4

Similarly, aflatoxins (AFs) are secondary metabolites produced by fungi Aspergillus, particularly flavus and parasiticus.5 There are four naturally occurring AFs, designated B1,B2, G1 and G2 with AFB1 the most common and toxic. The carcinogenic, mutagenic and immuno-suppressive effects of aflatoxins on several animals have been fully documented.6 Human liver cancer and levels of aflatoxins in the daily diet are strictly correlated as epidemiological studies.7 Furthermore, previous works show that aflatoxins levels are not reduced by domestic cooking with either microwave or conventional gas oven heating8 and that AFs do not decompose at the temperature of boiling water during the preparation of the drink.9 It is therefore important to have a simple and quantitative method of analysis to control levels of aflatoxins in food. In 2002 European Regulatory sets maximum limits for spices (Capsicum spp., Piper spp., Myristica fragrans, Zingiber officinale, Curcuma longa) (AFB15 g/kg; total AFs 10 g/kg) (02/472/EC). It is therefore imperative to ensure quality of the preparations derived from the traditional plants by using modern techniques and applying suitable standards.

Standardization of raw drugs in herbal industry is an important step towards quality control. Several analytical parameters such as physico–chemical constants, estimation of elements, heavy metals, microbial contamination, aflatoxins and pesticide residue are to be carried out as a measure of quality check. The World Health Organization10 emphasized certain quality standards and has proposed certain guidelines for the assessment and development of standard herbal products.

Acacia nilotica and Acacia leucophloea belongs to the family Leguminosae. It is a moderate sized tree much fissured, spines long, straight white, sharply prickled. A. nilotica flowers are yellow, fragrant in axillary and with globule heads. A. leucophloea flowers are creamy yellow, fragrant in axillary and with globule heads.11 The ethno medical information states that, the parts of the tree find use in diabetes, skin diseases and leucorrhoea. These are also used as an antidiarrheal, antidysenteric, antidiabetic. The stem bark is an astringent,12-17 used in ulcers and wound healing18,19 as an antidote to snake bite,20 in dysentery21 and for tooth ache.22 In the present paper an attempt was made for analysis of heavy metals pesticide residues, aflatoxins, microbial load and phytochemical assessment of A.nilotica and A.leucophloeaflowers.

MATERIALS AND METHOD

The fresh flowers of A.nilotica Linn, A.leucophloea Willd were collected from various domestic places in Thanjavur district, Tamilnadu, India, in the month of August 2009. The flowers were identified by Taxonomist, and authenticated by Prof.P.Jayaraman, Botanist, Director, Plant Anatomy Research Centre, Tambaram, Chennai, India. A voucher specimen number was PARC/2009/2093 and PARC/2009/2094 and it was deposited in CSMDRIA at Chennai for the future reference.

Reagent and Materials

Standard catechin, aflatoxins, was purchased from M/s Sigma chemicals. Aluminum plates precoated with silica gel 60 F254 of 0.2 mm thickness (E. Merck, Darmstadt, Germany) were used without pretreatment. All chemicals and solvents used were analytical and HPLC grade (E.Merck, Mumbai, India).

The procedures recommended in AOAC23 were followed to determine analysis of heavy metals, aflatoxins and pesticide residues. The powdered samples were weighed (each 1 g) into separate conical flasks and treated with 5 mL of concentrated nitric acid. The flasks were covered with watch glasses and heated for an hour; the contents of the flasks were treated with additional 5 mL of nitric acid, followed by 2 mL of 30% hydrogen peroxide solution. The heating was continued till the clear solution was obtained. The mixture was diluted with deionized water and filtered through Whatman No. 42 filter paper and the solutions were made up to 50 ml.24

Aflatoxins were determined by Kobra cell technique using Agilent HPLC instrument as per the method ASTA.25 Pesticide residues were analyzed using GC-MS Agilent instrument equipped with mass selective detector as per the method AOAC.23

For determination of microbial load, 1g of each sample was weighed accurately in separate flasks and 99 ml of sterile distilled water was added. The samples in the flask were kept in a mechanical shaker for few minutes to obtain uniform suspension of microorganisms. The dilution is 1:100 or 10-2 from, which 1 ml was transferred to 9 ml of sterilized distilled water to make a 1:1000 dilution and this procedure was repeated up to 10-6 dilution. Each 0.1 ml of serially diluted sample was inoculated to the sterile plates containing nutrient agar, SS agar and potato dextrose agar (PDA) by spread plate method.

Nutrient agar and SS agar plates were incubated at 37ºC for 24 hour and PDA plates were incubated at room temperature for 3-5 days. Bacterial and fungal colonies were counted using a colony counter.26

Thin layer chromatographic technique was used to separate the chemical compounds present in the drug. Various solvent systems were checked to separate the maximum number of chemical compounds in the drug. TLC of the methanolic extract developed in the mobile phase of Chloroform:Methanol (8:2 v/v). After air drying the plate was not visualized in UV 254 and 366 nm. The plate was then dipped in Vanillin -Sulphuric acid and heated in hot air oven at 105°C till the spots appeared.

RESULT AND DISCUSSION

The contents of heavy metals namely lead, mercury, cadmium and arsenic are found to be within the permissible limit27 for the A.nilotica and A.leucophloea, indicating that the flowers are safe to utilize as drugs (Table 1). The report for analysis of aflatoxins in the A.nilotica and A.leucophloea is given in the Table 3. The aflatoxins B1,B2, G1 and G2 were below the detecting level revealing that they are free from toxins and are safe for internal use. Further the studies indicated that the absence of these aflatoxins would help to increase in shelf life of the raw drug. The various pesticide residues such as α,β,γ,δ-hexachlorocyclohexane (α,β,γ,δ-HCH), o,p’-dichlorodiphenyl trichloroethane (o,p-DDT), p,p’-dichlorodiphenyltrichloroethane (p,p’-DDT), o,p-dichlorodiphenyl dichloroethylene (o,p-DDD), p,p-dichlorodiphenyldichloroethylene (p,p-DDD), α-endosulphan, β- endosulphan, o,pdichlorodiphenyldichloroethane (o,p-DDE) and p,p’-dichlorodiphenyldichloroethane (p,p-DDE) are analyzed. None of the above pesticides is detected (DL: 0.01 ppb) in the A.nilotica and A.leucophloea, indicating that they are safe for their usage as drugs (Table 2). Analysis for the microbial load for the A. nilotica and A.leucophloea is found to be within the limit of WHO guidelines, indicating that they are free from pathogens and can be used as drugs (Table 4).

The TLC analysis reveals that the marker compound Catechin was found to be present in Acacia nilotica flower extract but catechin was found to be absent in Acacia leucophloea flowers (Figure. 1). It is interesting to note that in stem bark of Acacia leucophloea the catechin was absent.28

Table 1

Heavy metal analysis results of A.nilotica Linn and A.leucophloea Willd flowers

HeavyMetalMaximum Permissible LimitObserved value

A.niloticaA.leucophloea
Arsenic (As)3 ppm0.002 ppm0.001 ppm
Cadmium (Cd)0.3 ppm0.25 ppm0.21 ppm
Mercury1 ppm0.12 ppm0.08 ppm
Lead (Pb)10 ppm0.16 ppm0.10 ppm
Table 2

Pesticide residue analysis results of A.nilotica Linn and A.leucophloea Willd flowers

PesticideDetection LimitA.niloticaA.leucophloea
Organo chlorine0.005 mg/kgBDLBDL
Organo phosphorus0.005 mg/kgBDLBDL
Table 3

Aflatoxin analysis results of A.nilotica Linn and A.leucophloea Willd flowers

AflatoxinStructureDetection LimitA.niloticaA.leucophloea
AF B1https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-721_img_1.jpg0.3 μg/kgBDLBDL
AF B2https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-721_img_2.jpg0.3 μg/kgBDLBDL
AF G1https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-721_img_3.jpg0.3 μg/kgBDLBDL
AFG2https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-721_img_4.jpg0.3 μg/kgBDLBDL
Table 4

Total microbial load results of A.nilotica Linn and A.leucophloea Willd flowers

ParametersA.niloticaA.leucophloea
Total Bacterial count3.3 × 10 3 cfu / g5.2 × 10 4 cfu / g
Total fungal count< 103 cfu / g3.3 × 10 2 cfu / g
Enterobacteriaceae< 10cfu / g< 10cfu / g
SalmonellaAbsentAbsent
S. aureusAbsentAbsent
Figure 1

TLC of A.nilotica Linn and A.leucophloea Willd flowers

https://s3-us-west-2.amazonaws.com/jourdata/pj/PharmacognJ-9-721_img_5.jpg

CONCLUSION

The flowers of Acacia nilotica and Acacia leucophloea exhibit a set of diagnostic characters, which will help to identify the drug in dried condition. It has been concluded from this study that estimation of heavy metals and pesticides residue, aflatoxin and total microbial load is highly essential for raw drugs or plant parts used for the preparation of single and compound formulation drugs. Plant-based traditional knowledge has become a recognized tool in search for new sources of drugs; it is clear that these herbal medicines will offer a platform for further research. So,concluding that both species (A.leucophloea and A. nilotica) can be distinguished on the basis of their secondary metabolites like catechin. Furthermore isolation of catechin is needed to explore its medicinal value in order to establish as a standard drug. Hence the periodic assessment is essential for quality assurance and safer use of herbal drugs.

Acknowledgements

We are thankful to the Director General, CCRAS,M/O AYUSH,GOI,New Delhi for the facilities provided.

Notes

[1] Conflicts of interest CONFLICT OF INTEREST We declare that we have no conflict of interest.

REFERENCES

1 

Chan K. , editor. Some aspects of toxic contaminants in herbal medicines. Chemosphere. 2003;52(9):1361–71

2 

Miraldi E, Giachetti D, Ferri S , authors. Quality control of aromatic drugs reported in European Pharmacopoeia 3rdedition. Il Farmaco. 2001;56(5):365–71

3 

Tewary DK, Kumar V, Shanker A , authors. Leaching of pesticides in herbal decoction. Chemical Health and Safety. 2004;11(4):25–9

4 

Sankararamakrishnan N, Sharma AK, Sanghi R , authors. Organochlorine and organophosphorous pesticide residues in ground water and surface waters of Kanpur, Uttar Pradesh, India. Environment International. 2005;31(1):113–20

5 

Reddy SV, Kiran Mayi D, Uma Reddy M, Thirumala-Devi K, Reddy DV , authors. Aflatoxins B1 in different grades of chillies (Capsicum annum L.) in India as determined by indirect competitive-ELISA. Food Additives and Contaminants. 2001;18(6):553–8

6 

IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. World Health Organization, International Agency for Research on Cancer. 1993;

7 

Groopman JD, Kensler W , authors. Temporal patterns of aflatoxin albumin adducts in hepatitis B surface antigen-positive and antigen negative residents of Daxin, Qidong County, People’s Republic of China. Cancer Epidemiology Biomarkers and Prevention. 1996;5(4):253–61

8 

Midio AF, Campos RR, Sabino M , authors. Occurrence of aflatoxins B1, B2, G1 and G2 in cooked food components of whole meals marketed in fast food outlets of the city of Sao Paulo, SP, Brazil. Food Additives and Contaminants. 2001;18(5):445–8

9 

Feuell Aj , author. Aflatoxin in groundnuts. Part 9: Problems of detoxification. Tropical Science. 1996;8:61–70

10 

Mudaliyar CS , author. Siddha Materia Medica. Part I, Chennai:. Medicinal Plants division Department of Indian Medicine and Homeopathy. 2002;6:381–6

11 

Ravindrasharma , author. Medicinal plants of India-An encyclopedia. 2003;4:

12 

Ahluwalia KS , author. Medicinal plants of Kerala-IV. Nagarjun. 1968;11:300–3

13 

Yoganarasimhan SN, Bhatt AV, Togunashi VS , authors. Medicinal plants from Mysore district Karnataka. Indian Drugs Pharmaceut Ind. 1979;14:7–22

14 

Yoganarasimhan SN, Nair KV, Keshavamurthy KR, Govindaiah , authors. Medico botany of Karnataka-3. Utilization of floristic wealth for the economic development of Kanakapura Taluk, Bangalore district. J Econ Tax Bot. 1982;6:97–108

15 

Kapoor SL, Kapoor LD , authors. Medicinal plant wealth of the Karimnagar district of Andhra Pradesh. Bull Med Ethnobot Res. 1980;1:120–144

16 

Sebastian MK, Bhandari MM , authors. Medico-ethno botany of Mount Abu, Rajasthan, India. J Ethnopharmacol. 1984;12(12):223–30

17 

Khan MA, Khan T, Ahmad Z , authors. Barks used as source of medicine in Madhya Pradesh, India. Fitoterapia. 1994;65:444–6

18 

Apparnantham T, Chelladurai V , authors. Glimpses of folk medicines of Dharmapuri forest division, Tamilnadu. Ancient Sci Life. 1986;5:182–5

19 

Reddys SV, Kiran MD, Uma RM, Thirumala D, Reddy K, V.R. D. , authors. Aflatoxins B1 in different grades of chillies (Capsicum annum L) in India as determined by indirect comp. 2001;12:

20 

Selvanayagam ZE, Gnavanendhan SG, Balakrishna K, BhimaRao R, UsmanAli S , authors. Survey of medicinal plants with antisnake venom activity in Chengalpattu district, Tamilnadu, India. Fitoterapia. 1995;66(6):488–92

21 

Hemadri K, Raj PV, Rao SS, Sharma CRR , authors. Folklore claims from Andhra Pradesh-I. J Sci Res Plant Med. 1980;1:37–49

22 

Balaji RNS, Rajasekhar D, Chengal RD, Nagaraju N , authors. Ethno-medicinal notes on some plants of tirumala hills for dental disorders. Ethnobotany. 1996;8:88–91

23 

Horwitz W, Latimer GW , authors. Official Methods of Analysis of AOAC International.Chapter 10.18th ed. Maryland:: AOAC International; p. 18–23

24 

Sahito SR, Kazi TG, Kazi GH, Jakhrani MA, Shaikh MS , authors. Trace elements in two varieties of Indigeneous Medicinal Plant Catharanthus roseus. Sciences. 2001;1:74–7

25 

Official Analytical Methods of the American Spice Trade Association. (New Jersey:). ASTA, Inc. 1997;4:149–52

26 

World Health Organization;. Quality Control Methods for Medicinal Plant Materials.Geneva: WHO Publications; 1998. p. 10–31

27 

The Ayurvedic Pharmacopoeia of India, Part II. New Delhi: Ministry of Health and Family Welfare. 2008. p. 168

28 

Sulaiman CT, Gopalakrishnan K , authors. Comparative phytochemical studies in Selected Acacia Species. International Journal of Pharmacy and Pharmaceutical Sciences. 2012;4(5):458–60